3,3′-diindolylmethane (DIM), and its analogues and derivatives have a broad spectrum of biological activities for which reason DIM may be regarded as a pharmacologically active compound of great promise. 3,3-diindolylmethane (DIM) is the main oligomer product of indole-3-carbinol (I3C) proved to be highly selective is respect of transformed cells of varied origin (Aggarwal B. B., Ichikawa H. (2005), Molecular Targets and Anticancer Potential of Indole-3-Carbinol and Its Derivatives, Cell Cycle, 4(9), 1201-1215). Pharmacokinetic studies have shown that perorally administered I3C is almost immediately transformed to DIM in the acidic medium of the stomach (Arneson D. W., Hurwitz A., McMahon L. M., Robaugh D. (1999), Presence of 3,3′-Diindolylmethane in Human Plasma after Oral Administration of Indole-3-Carbinol (Abstr.), Proc. Am. Assoc. Cancer Res., 40, 2833). Many researchers studying anticancer activity of I3C tend, therefore, to accept the idea that a majority of clinical effects registered upon its administration are actually produced by the dimer form of indole-3-carbinol, or DIM.
It has been demonstrated experimentally that nearly all multiple anticancer mechanisms induced by I3C in vitro and in vivo are inherent in DIM as well (Chang X., Tou J. C., Hong C., et al. (2005), 3,3′-Diindolylmethane Inhibits Angiogenesis and the Growth of Transplantable Human Breast Carcinoma in Athymic Mice, Carcinogenesis, 264(4), 771-778; Firestone G. L., Bjeldanes L. F. (2003), Indole-3-Carbinol and 3,3-Diindolylmethane Anti-Proliferative Signaling Pathways Control Cell Cycle Gene Transcription in Human Breast Cancer Cells by Regulating Promoter-Sp1 Transcription Factor Interactions, J. Nutr., 133, 2448S-2455S; Ge X., Yannai S., Rennert G., et al. (1996), 3,3′-Diindolylmethane Induces Apoptosis in Human Cancer Cells, Biochem. Biophys. Res. Commun., 228, 153-158; Hong C., Kim H. A., Firestone G. L., et al. (2002), 3,3′-Diindolylmethane (DIM) Induces a Cell Cycle Arrest in Human Breast Cancer Cells That Is Accompanied by Sp-1-Mediated Activation of p21 WAF1/CIP1 Expression, Carcinogenesis, 23, 1297-1305; Leibelt D. A., Hedstrom O. R, Fisher K. A. (2003), Evaluation of Chronic Dietary Exposure to Indole-3-Carbinol and Absorption Enhanced 3,3′-Diindolylmethane in Sprague-Dawley Rats, Toxicol. Sci., 74, 10-21; Li Y., Li X., Sarkar F. H. (2003), Gene Expression Profiles of I3C- and DIM-Treated PC3 Human Prostate Cancer Cells Determined by cDNA Microarray Analysis, J. Nutr., 133, 1011-1019; Nachshon-Kedmi M., Yannai S., Haj A., Fares F. A. (2003), Indole-3-Carbinol and 3,3′-Diindolylmethane Induces Apoptosis in Human Prostate Cancer Cells, Food Chem. Toxicol., 41, 745-752). This conclusion applies to prostate cancer as well. Like I3I, DIM in vitro and in vivo inhibits growth of prostate cancer cells (Li Y., Li X., Sarkar F. H. (2003), Gene Expression Profiles of I3C- and DIM-Treated PC3 Human Prostate Cancer Cells Determined by cDNA Microarray Analysis, J. Nutr., 133, 1011-1019; Nachshon-Kedmi M., Fares F. A., Yannai S. (2004), Therapeutic Activity of 3,3′-Diindolylmethane on Prostate Cancer in an in vivo Model, Prostate, 61 (2), 153-160) and induces their apoptosis (Li Y., Li X., Sarkar F. H. (2003), Gene Expression Profiles of I3C- and DIM-Treated PC3 Human Prostate Cancer Cells Determined by cDNA Microarray Analysis, J. Nutr., 133, 1011-1019; Nachshon-Kedmi M., Yannai S., Fares F. A. (2004), Induction of Apoptosis in Human Prostate Cancer Cell Line, PC3, by 3,3′-Diindolylmethane Through the Mitochondrial Pathway, Br. J. Cancer, 91, 1358-1363), in which case it, similarly to I3C, displays its activity at the submolecular level by regulating the expression of genes responsible for proliferation, differentiation, and survivability processes (Li Y., Li X., Sarkar F. H. (2003), Gene Expression Profiles of I3C- and DIM-Treated PC3 Human Prostate Cancer Cells Determined by cDNA Microarray Analysis, J. Nutr., 133, 1011-1019) and inhibiting multiple signaling pathways leading to cellular hyperproliferation.
The hormone-sensitive prostate cells (culture LNCaP) have been used to demonstrate that DIM can be bound concurrently to androgen receptors to suppress in this way their translocation into the nucleus and successive activation of gene transcription, and also expression of the gene promoter encoding the prostate-specific PSA antigen. The PSA protein (specific prostate protease) is a classical marker of prostate cancer that is produced and secreted in abundance by prostate cancel cells. The same paper established, after structural studies undertaken, that DIM is very similar in molecular geometry to the well-known synthetic anti-androgen Casodex (Le H. T, Schaldach C. M., Bjeldanes L. F. (2003), Plant-Derived 3,3′-Diindolylmethane Is a Strong Androgen Antagonist in Human Prostate Cancer Cells, J. Biol. Chem., 278, 21136-21145) that, in contrast to DIM, however, promotes translocation of androgen receptors into the nucleus (Masiello D., Cheng S., Bubley G. J., et al. (2002), Bicalutamide Functions as an Androgen Receptor Antagonist by Assembly of a Transcriptionally Inactive Receptor, J. Biol. Chem., 277, 26321-26326).
The capacity of DIM to display anti-angiogenic activity, discovered only recently, is an extremely significant development. Pathological growth of vessels almost always accompanies hyper- and neoplastic processes. It is common knowledge that unless a network of capillary vessels is formed to supply oxygen and nutrients to a new tumor of 1 to 2 mm in diameter the tumor would not continue to grow at all. It has been demonstrated that micromolar concentrations of DIM in vitro suppress proliferation and migration of endothelial cells and their capacity to form vessels effectively. In vivo, DIM injected subcutaneously to experimental animal (5 mg/kg daily) was 74% effective in suppressing pathological neoangiogenesis (Chang X., Tou J. C., Hong C., et al. (2005), 3,3′-Diindolylmethane Inhibits Angiogenesis and the Growth of Transplantable Human Breast Carcinoma in Athymic Mice, Carcinogenesis, 264(4), 771-778; McCarty M. F., Block K. I. (2005), Multifocal Angiostatic Therapy: An Update, Integrative Cancer Therapies, 4(4), 301-314).
The nuclear transcription factor NF-κB is the most significant molecular target displaying an activity that modern target preparations (directional preparations) developed and adopted in clinical practice are intended to block. It has been proved that this factor mediates the inflammatory response and has a key role in regulating proliferative (anti-apoptotic), angiogenic, migratory, and invasive cellular activities at the final stage of signaling pathways induced by growth factors and cytokines. Moreover, translocation of the active factor into the nucleus and transcription activation of genes responsible for these processes is a significant event. It has been found that, if used in vitro, DIM (Rahman K. M., Ali S., Aboukameel A., et al. (2007), Inactivation of NF-KappaB by 3,3′-Diindolylmethane Contributes to Increased Apoptosis Induced by Chemotherapeutic Agent in Breast Cancer Cells, Mol. Cancer Ther., 6(10), 2757-2765; Rahman K. M., Sarkar F. H. (2005), Inhibition of Nuclear Translocation of Nuclear Factor-κB Contributes to 3,3′-Diindolylmethane-Induced Apoptosis in Breast Cancer Cells, Cancer Res., 65, 364-371) and its metabolic predecessor I3C are effective in suppressing nuclear translocation and activity of NF-κB. This means that, in addition to its anti-proliferative and anti-angiogenic effect, the DIM-base preparation is capable of suppressing local inflammatory reactions that frequently attend hyper- and neoplastic processes in hormone-dependent organs and tissues.
A detailed study of patients with regression of cervical dysplasias conducted within the framework of placebo-controlled clinical research has helped trace a direct link between the positive dynamics of the disease and the efficiency of I3C conversion to DIM (Sepkovic D. W., Bradlow H. L., Bell M. (2001), Quantitative Determination of 3,3′-Diindolylmethane in the Urine of Individuals Receiving Indole-3-Carbinol, Natr. Cancer, 41, 57-63). The high DIM concentration was determined in the urine of patients receiving the preparation.
One of the most recent experimental studies demonstrated the capacity of DIM to induce apoptosis of human cervical HPV-infected keratinocytes in vitro. Moreover, in one of the three cellular cervical cancer lines studied, DIM displayed a much higher efficiency than I3I. The value of LD50 was 50 to 60 μM for DIM and 200 μM for I3C, respectively, but, unlike its metabolic predecessor (I3C), DIM did not induce any apoptotic changes in normal (untransformed) keratinocytes (Chen D. Z., Qi M., Auborn K., Carter T. H. (2001), Indole-3-Carbinol and Diindolylmethane Induce Apoptosis of Human Cervical Cancer Cells and in Murine HPV16-Transgenic Preneoplastic Cervical Epithelium, J. Nutrit., I3I, 3294-3302).
To conclude, DIM has been discovered recently to have yet another property, perhaps one of its most important advantages—its immunomodulating activity. The researchers have shown that when used in vitro DIM stimulates IFNγ-dependent signaling pathways in tumor cells by activating expression of IFNγ receptors, and also other IFN-responsive regulatory proteins.
The peroral method of dosing DIM-base preparations must be given preference because it offers a series of advantages over other dosing methods, in particular, patient comfort, flexible treatment tactics, and treatment costs. Peroral dosing, however, limits significantly the biological availability of DIM because of its poor solubility and low absorption efficiency in the small intestines. DIM typically shows poor solubility in physiological salt solutions and has a limited capacity to pass through barrier membranes. Furthermore, this compound is known to be bound to blood plasma proteins and be involved in various unspecific reactions in the bloodstream that reduce greatly the efficiency of its delivery to the disease focus.
Several pharmaceutical compositions based on pegylated vitamin E (TPGS) have been developed recently as a way to dispose of the above-mentioned problems (Anderton M. J., Manson M. M., et al. (2004), Physiological Modeling of Formulated and Crystalline Diindolylmethane Pharmacokinetics Following Oral Administration in Mice, Drug Metabolism and Disposition, 32(6), 632-638). Pegylated vitamin E is known for its capacity to enhance solubility of various compounds in water (Constantinides P. P., Tustian A., Kessler D. R. (2004), Tocol Emulsions for Drug Solubilization and Parenteral Delivery, Adv. Drug Deliv. Rev., 56, 1243-1255) and improve their biological availability when administered perorally (Wu S. H. W., Hopkins W. K. (1999), Characteristics of d-α-Tocopheryl PEG 1000 Succinate for Applications as an Absorption Enhancer in Drug Delivery Systems, Pharm. Technol., 23, 52-68). TPGS-base compositions, however, can increase insignificantly only (by 50% to 100% only) the biological availability of DIM, and its analogues and derivatives (Zeligs, et al., U.S. Pat. No. 6,416,793, Formulation and Use of Controlled-Release Indole Alkaloids), for which reason the therapeutic potential of these compounds cannot be utilized in full.